The present invention relates generally to the field of structural genomics and, more specifically, to the use of crystallography to examine protein crystals for genomic research.
In biological research, particularly in the field of genomics, crystallography is a tool used to examine the characteristics of proteins. However, such proteins are typically developed in a liquid medium, and therefore must be crystallized in an orderly fashion before detailed crystallography techniques may be used. A typical method for crystallizing such proteins is through vapor diffusion. In a method known as the xe2x80x9changing dropxe2x80x9d method, a well solution is placed in the many separate sample wells of the sample tray. For each sample well, a drop of protein liquid is applied to a slide, which is placed over the sample well with the drop hanging down toward the well solution. Because of different relative concentrations of the well solution and the droplet solution, over time, liquid diffuses out of the droplet and into the sample well, resulting in the crystallization of the protein on the surface of the slide.
Depending on the conditions under which the crystallization process takes place, the formation of a crystal may take anywhere from hours to months. While some crystals are visible to the naked eye, the sample slides must usually be examined with a microscope one at a time to determine whether protein crystallization has taken place. Of course, for those protein samples that have not yet crystallized, the slides must be reexamined on a regular basis until the crystallization is observed. For a relatively large number of samples, this is obviously a long and labor-intensive process.
In accordance with the present invention, a screening apparatus is provided for monitoring crystal formation in a crystal growth medium that makes use of an x-ray source and detector. X-ray energy from the x-ray source is incident on a sample container and undergoes diffraction if in the presence of a crystal structure. Any such diffracted x-ray energy is detected by the x-ray detector, the output of which is indicative of the presence or absence of such a crystal structure. In this way, one may determine whether any significant crystal formation has taken place in the crystal growth medium, without the need for visual examination of the sample container. This is particularly useful for the examination of biological crystal formation common in genomics research.
In a preferred embodiment, the screening apparatus also includes a positioning apparatus for locating the sample container relative to the x-ray source and x-ray detector. The positioning apparatus has a support that is remotely movable in at least two dimensions, allowing the precise positioning of the sample container relative to the x-ray source and detector. This is particularly useful in the preferred embodiment of the invention, in which the sample container is one of a plurality of sample containers each having a separate crystal growing medium. The sample containers may be part of a contiguous array, such as in a sample tray having an array of sample wells. In such a case, the positioning apparatus may be used to move the sample containers so as to position them sequentially relative to the x-ray source and detector, thereby allowing sequential examination of the sample containers. In addition, the source and detector may be arranged to operate in reflective mode or in transmission mode. If used in transmission mode, the positioning apparatus preferably has an open section located between the source and a sample well under investigation so as to not interfere with the source x-ray energy.
The x-ray source and detector may be arranged such that the exposure of x-rays from the source covers a two-dimensional area of the sample container being examined, in particular, an area over which any significant crystal formation would be expected to appear. The detector, similarly, is a two-dimensional detector, providing simultaneous detection of x-ray energy diffracted from a similar two-dimensional region of the sample container. Therefore, a simultaneous set of pixel intensities may be collected that is indicative of any presence of crystal structures across the two-dimensional area of the sample container under investigation.
A control apparatus is preferably used to control the various aspects of the screening apparatus, including the triggering of the x-ray source and the collection and processing of data from the detector. The control apparatus may also be used to control the positioning apparatus to synchronize the alignment of the various sample containers in an array with the operation of the x-ray source and detector. In this way, a the system may be used to automatically analyze the entire array of sample containers to determine which, if any, show the formation of any significant crystal structure.
In addition to the structural aspects of the invention, various techniques are also provided that may be used to evaluate the intensity data from the pixels of the detector to make a determination of whether or not a crystal is present. One such technique involves determining the number of pixels having an intensity level exceeding a minimum pixel intensity level and comparing that number to a predetermined minimum number selected as being indicative of the presence of said crystal structure. In another method, the outputs from a predetermined number of pixels having the highest intensity levels are averaged and compared to an overall average intensity value of all the pixels. In yet another method, the pixel intensity values that are indicative of the presence of a crystal peak in the detected spectrum are isolated and integrated. This integrated crystal peak intensity is then compared to an integrated intensity of all the detector pixels.
A particular type of sample container may be used with the present invention to minimize scattering from the well solution despite the x-ray source being on one side of the container while the detector is on the opposite side. The solution is retained within a reservoir region of the container, and the sample is located at a sample location. However, the sample location is arranged relative to the reservoir such that a beam of x-ray energy travels from the x-ray source to the sample location without being incident on the well solution.
In a first version of this embodiment, the sample is located on a platform and is retained there under the force of gravity. Although the well solution resides at a lower point in the sample container than the sample, a region below the platform is devoid of well solution, and the x-ray beam passes through this region and through the platform to reach the sample. The platform is preferably smaller within a portion of it through which a maximum portion of x-ray energy passes (typically in the center), than in surrounding portions. The surface of the platform upon which the sample resides may be concave, as may be the opposite surface. Preferably, the material of the platform is mostly or entirely amorphous.
In another variation of this embodiment, the sample resides on the underside of a surface within the sample container under the force of surface tension. Again, despite the well solution being at a lower point within the sample container, a region below the sample is clear of any well solution. Preferably, a baffle surrounds this portion of the container, and excludes the well solution therefrom. Although the well solution is still in the same enclosed space with the sample, an x-ray beam may be directed through the portion of the container below the sample without encountering any well solution.